Assembly dynamics of PML nuclear bodies in living cells

Abstract

The mammalian cell nucleus contains a variety of organelles or nuclear bodies which contribute to key nuclear functions. Promyelocytic leukemia nuclear bodies (PML NBs) are involved in the regulation of apoptosis, antiviral responses, the DNA damage response and chromatin structure, but their precise biochemical function in these nuclear pathways is unknown. One strategy to tackle this problem is to assess the biophysical properties of the component parts of these macromolecular assemblies in living cells. In this study we determined PML NB assembly dynamics by live cell imaging, combined with mathematical modeling. For the first time, dynamics of PML body formation were measured in cells lacking endogenous PML. We show that all six human nuclear PML isoforms are able to form nuclear bodies in PML negative cells. All isoforms exhibit individual exchange rates at NBs in PML positive cells but PML I, II, III and IV are static at nuclear bodies in PML negative cells, suggesting that these isoforms require additional protein partners for efficient exchange. PML V turns over at PML Nbs very slowly supporting the idea of a structural function for this isoform. We also demonstrate that SUMOylation of PML at Lysine positions K160 and/or K490 are required for nuclear body formation in vivo.We propose a model in which the isoform specific residence times of PML provide both, structural stability to function as a scaffold and flexibility to attract specific nuclear proteins for efficient biochemical reactions at the surface of nuclear bodies.MCS code: 92C37

Dynamic Response of IFI16 and Promyelocytic Leukemia Nuclear Body Components to Herpes Simplex Virus 1 Infection

“…4A). These results are consistent with the previous FRAP experiments (11,(35)(36)(37). By applying this technique to cells at the edge of developing ICP0-null mutant HSV-1 plaques, characteristic recruited patterns of hDaxx and PML could be seen via the mCherry fluorescence.…”

“…Previous work using fluorescence recovery after photobleaching (FRAP) had demonstrated that hDaxx is highly mobile in the nucleus, with an exchange rate between PML NBs and the general nucleoplasm measured in terms of seconds, while that for PML was slower but still in the low numbers of minutes (11,(35)(36)(37). It had previously been suggested that this dynamic behavior would enable the formation of novel PML NB-like foci in association with HSV-1 genomes without the need for movement of preexisting PML NBs (11 vectors were constructed that expressed either hDaxx or PML linked to a fusion protein that included mCherry and a photoactivatable version of EGFP (30).…”

PML isoforms I and II participate in PML-dependent restriction of HSV-1 replication

“…However, our data are consistent with a previous finding of increased recruitment of hDaxx into foci containing overexpressed PML.VI (Block et al, 2006). The efficient recruitment of Sp100, hDaxx and ATRX into PML.V foci is consistent with the suggestion that this isoform plays a structural role in nucleating ND10 (Brand et al, 2010;Weidtkamp-Peters et al, 2008). Another surprising feature of these results was that expression of PML.I in HepaRG cells appeared to disturb normal ND10 integrity, because colocalisation with hDaxx and ATRX was poor even in the PML-positive background ( Fig.…”

“…It has been proposed that PML.V plays a structural role in nucleating ND10 (Brand et al, 2010;Weidtkamp-Peters et al, 2008), but it is also important to consider how the isoform-specific sequences might affect partner recruitment and exchange. Thus, the strong colocalisation of PML.V, PML.VI and PML.I.7a with Sp100, hDaxx and ATRX could be influenced not only by the efficiency of recruitment of these other ND10 proteins, but also by the dynamic stability of that recruitment.…”

Herpes Simplex Virus 1 Ubiquitin Ligase ICP0 Interacts with PML Isoform I and Induces Its SUMO-Independent Degradation

“…We conclude that the ICP0-PML.I interaction reflects a countermeasure to PML-related antiviral restriction. P romyelocytic leukemia protein nuclear bodies (PML-NBs), also known as ND10, are dynamic punctuate structures within the nuclei of mammalian cells that harbor a large number of permanently or transiently localized proteins (8,22,44). ND10 have been associated with many cellular functions, including DNA repair (17), regulation of transcription (42, 60), chromatin assembly and modification (18, 32), apoptosis (1, 55), stress (39), senescence (3), the ubiquitin pathway (30, 35, 36,) and oncogenesis (47, 48; reviewed in reference 2).…”